DESCRIPTION (adapted from the investigator's summary): The presence of seven compatible plasmids in Bacillus megaterium QM B1551 provides a unique system in which to study plasmid maintenance, replication regulation, genes and possible plasmid interactions. This work will provide the framework for future studies on the role of plasmids in B. megaterium and lead to the development of more stable, compatible replicons for gram-positive cloning Moreover, among the bacilli, clostridia, streptococci, and staphylococci are many pathogenic species that harbor plasmids with genes for virulence factors. Are these plasmids ever transferred to the nonpathogenic species in the soil? How extensive is their distribution and host range? Five replicons from the plasmids have been cloned and four have been sequenced. Several plasmid libraries have been constructed and new phenotypes have been found to be plasmid-borne. Based on this foundation , the specific aims of the proposal are: to initiate a study to determine the host range, homology with other plasmids and distribution of clone I in Bacillus and other gram positive bacteria by hybridizations and transformations. This will include an investigation of a possible pathogenic isolate. 2. To clone DNA flanking the functional clone I replicon, determine the source plasmid and identify additional genes that may contribute to stability, partitioning and copy number by subcloning and deletion analysis. 3. To establish the type and direction of replication by 2-D gel electrophoresis and electron microscopy. 4. To determine the nature, role and regulation of the putative rep gene and investigate the ability of the Rep protein to interact with its unusual iteron region. This will be done by northerns, primer extensions and gel mobility assays. This project should produce significant data on plasmid replication, transfer, evolution and gene function among the gram positives and may be the first broad survey of theta replicons and their first in-depth study. Since no significant homology to QM B1551 replicon proteins or genes has been found in GENE bank this characterization of seven plasmids in B. megaterium could be the key to investigating plasmids that have not been isolated or sequenced as yet.